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Optimization of urinary sample pre paration for proteomic analysis in kidney disease diagnosis
Juránková, Lada ; Přikryl, Petr (advisor) ; Pacáková, Věra (referee)
This work is focused on urinary sample preparation before their proteomic analysis by liquid chromatography with mass spectrometric detection. Urinary proteomics is in many cases inefficient due to low urine protein concentration, variations in pH and the presence of high amount of salts, pigments, and other metabolites. These components can interfere with proteomic protocol and with subsequent mass spectrometric analysis. Therefore, there is the need to develop new effective sample preparation method. The main benefit of this work is the optimization of the method for the effective preconcentration of proteins from urine samples using carboxylate-modified paramagnetic microparticles and the principles of HILIC chromatography. This innovative process was compared with a routine method using centrifugal filters (FASP) and the method based on proteins isolation by divinyl sulfone activated cellulose magnetic particles. Using carboxylate paramagnetic microparticles, a total of 856 proteins was identified from only 0,5 mL of healthy human urine by LC-MS/MS. This optimized protocol provided comparable results to the standard FASP method in a much shorter time and with a lower cost per analysis.
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Monitoring of selected steroid hormones by HPLC-MS/MS and studying their impact on mouse sperm in vitro.
Bosáková, Tereza ; Komrsková, Kateřina (advisor) ; Pacáková, Věra (referee)
The present project aims were development of a new sensitive separation method using high performance liquid chromatography with tandem mass spectrometric detection, which enables quantification of selected steroid hormones, namely 17α-ethynylestradiol (EE2) and progesterone (PRG) in M2 fertilizing medium. M2 medium is used during sperm capacitation in vitro, which is the physiological process of complex molecular and biochemical changes, essential for obtaining fertilizing ability of male gametes. Optimized separation and detection conditions (column Kinetex EVO C18 with mobile phase consisted of 50/50 (v/v) acetonitrile/water with 0.1% formic acid in both components) were applied on monitoring of concentration changes of each hormone during time dependent sperm capacitation. The concentration tested for EE2 were 200, 20 and 2 µg/l and for PRG 200 µg/l only. It was found that concentration of free, unbounded EE2 firstly decreases, reaches the minimum in about 60 min of capacitation and then increases again. This trend was observed for all three tested concentrations. Concentration (200 µg/l) of free unbounded PRG decreased during whole capacitation. The following kinetic analysis will be based on the concentration dependences of the free unbound hormones on the capacitation time. The results of...
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Analytical chemistry unravels the diversity and function of chemicals used for communication and defence in termite societies
Krasulová, Jana ; Bosáková, Zuzana (advisor) ; Pacáková, Věra (referee) ; Lapčík, Oldřich (referee)
Analytical chemistry plays a crucial role in studies on chemical ecology and only the development of sophisticated methods enables the detection of biologically active compounds that are usually present in minute quantities and often in very complex mixtures. My thesis is dedicated to the application of modern analytical techniques and instrumentation to unravel the identity, chemical diversity and function of semiochemicals and defensive compounds used by various species of termites. The first section of this thesis aims at the identity of chemicals used in communication, the pheromones. I studied the chemistry of the trail-following communication in three selected species of termites. Besides the identification of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol, the most frequent termite trail-following pheromone, as the trail-following pheromone in Psammotermes hybostoma (Rhinotermitidae), I participated in the description of two new structures, (10Z,13Z)-nonadeca-10,13-dien-2-one in Glossotermes oculatus (Serritermitidae) and syn-4,6-dimethylundecan-1-ol in Hodotermopsis sjoestedti (Archotermopsidae). We identified (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol to be the female sex pheromone in Psammotermes hybostoma, and syn-4,6-dimethylundecanal to be the male sex pheromone in Hodotermopsis sjoestedti. I also...
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Determination of non-esterified fatty acids in plasma
Lacinová, Petra ; Pacáková, Věra (advisor) ; Staňková, Barbora (referee)
The determination of non-esterified fatty acids (NEFA) in plasma is possible by many routine biochemical methods. For more detailed metabolic studies it is required to analyse the fatty acid (FA) profile. The NEFA profile determination is usually performed by the capillary gas chromatography (GC), but the sample preparation is not uniform. NEFA may be isolated by the extraction process or by the preparative thin-layer chromatography (TLC). The aim of this study was to compare these two separation procedures. The samples of pooled plasma from volunteer donors were analyzed by capillary GC after previous separation procedure by the TLC and the liquid-liquid extraction. The results were compared by the t-test for both the absolute concentration of individual FA provided by the internal standard (margaric acid) method and the relative abundance (Rel%). The reproducibility of the results was significantly better for the liquid-liquid extraction method than the TLC. The relative standard deviations (RSD) of the FA groups (unsaturated, monosaturated and polysaturated) were from 4.3 to 11.3 % vs. 8.6 to 33.8 % for the relative abundance and from 16.5 to 25.5% vs. 15.4 to 47.4% for the absolute concentration. For each FA, which were represented by more than 0.1 Rel %, RSD ranged from 4.5 to 47.8% vs. 9.5 to...
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Determination of pepsinogens using IgY and IgG
Kulhavá, Lucie ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...
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Development of HPLC-DAD-MS/MS method for determination of selected toxins in Amanita phalloides.
Musil, Karel ; Bosáková, Zuzana (advisor) ; Pacáková, Věra (referee)
(EN) This work has been focused on the development and optimization of analytical methodology, using high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography with diode array and tandem mass spectrometric detection (HPLC-DAD-MS/MS) for the determination of amatoxins (α- and β-amanitin) and phalotoxins (phallacidin and phalloidin) in the crude extract from cap of Amanita phalloides with the option to later use for clinical purposes. In order to guarantee the reliability of the analytical results the influence of various parameters on the quality of the separation and the determination of toxins was studied. The developed HPLC-DAD method achieves good linearity in the concentrations range 1 - 100 µg/ml, with correlation coefficients higher than 0,997. Limits of detection (LOD) and quantitation (LOQ) were calculated for all the studied toxins with following values: for α-amanitin 0,90 µg/ml (LOD); 2,99 µg/ml (LOQ), β-amanitin 1,07 µg/ml (LOD); 3,56 µg/ml (LOQ), phallacidin 2,17 µg/ml (LOD); 7,26 µg/ml (LOQ) and phalloidin 0,79 µg/ml (LOD); 2,64 µg/ml (LOQ). Key words: Amanita phalloides,-amanitin,-amanitin, phallacidin, phalloidin, solid-liquid extraction, HPLC-DAD, HPLC-DAD-MS/MS .
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Peptide inhibitors immobilized on magnetic particles and Sepharose used for separation of stomach aspartate proteinases
Rajčanová, Michaela ; Kučerová, Zdenka (advisor) ; Fusek, Martin (referee) ; Pacáková, Věra (referee)
IN ENGLISH Human gastric juice contains mainly aspartate proteinases: pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens and they are activated to the corresponding pepsins in the acidic environment of the gastric lumen. The levels of pepsinogens in serum reflect the morphological and functional status of gastric mucosa. A subject of this thesis is a part of a long-term investigation that focuses on the elaboration of methods for separation gastric aspartate proteainases that would be suitable for diagnostic purposes. The preparation of new type ligands was a concrete subject of PhD. thesis that after their immobilization they can enable the separation of aspartate proteinases. Four heptapeptides containing D-leucinyl residue were synthetized (Val-D-Leu-Pro-Phe-Phe-Val- D-Leu, Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu and Val-D- Leu-Pro-Phe-Tyr-Val-D-Leu. The prepared heptapeptides immobilized on agarose magnetic particles were used for the study of their interaction with porcine pepsin A and rat pepsin C. While porcine pepsin A was adsorbed to all heptapeptides immobilized to magnetic particles, rat pepsin C was not retarded. Similar results were obtained using heptapeptides immobilized to Sepharose. The situation was more complicated...
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Modern Trends in High Performance Liquid Chromatography and Their Application
Kozlík, Petr ; Bosáková, Zuzana (advisor) ; Pacáková, Věra (referee) ; Cajthaml, Tomáš (referee)
(EN) The dissertation thesis is focused on major trends in high performance liquid chromatography such as miniaturization of separation systems in hyphenation with high- sensitivity detection or characterization of new types of stationary phases for the separation of polar compounds in systems suitable for mass detection. Application of recently developed stationary phases in hydrophilic interaction liquid chromatography (HILIC) is also considered. Capillary liquid chromatography with tandem mass spectrometry (cLC-MS/MS) method was developed for determination of five estrogenic pollutants in samples of water. Several new sorption materials for solid phase extraction (SPE) were compared to obtain sufficient recovery of all the tested analytes. Discovery DSC-18Lt column provided the highest recovery (95 - 100 %). The optimized cLC-MS/MS with SPE was used for determination of estrogens in water samples in the order of units to tens of ng/L. HILIC separation systems with silica gel, cyclofructan and isopropyl cyclofructan modified silica stationary phases were tested and compared. Ability to donate protons and dispersion interactions are the main interactions that affect retention in HILIC with cyclofructan-based columns while they are less important in separation systems with bare silica stationary...
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Enantioseparation of selected FMOC-derivatized amino acids on Chirobiotic T a Chiralpak QD-AX columns.
Kašpárková, Marie ; Bosáková, Zuzana (advisor) ; Pacáková, Věra (referee)
In this work, an enantioselective HPLC method with fluorimetric detection (λEx = 254 nm, λEm = 314 nm) was developed and subsequently optimized for chiral separation of four amino acids (D/L-alanine, D/L-valine, D/L-leucine, D/L-isoleucine) in derivatized form with an emphasis on enantioseparation of D-analogues. Derivatization of amino acids was performed using 9-fluorenylmethyl chloroformate (FMOC-Cl). Retention and enantioseparation behavior of studied analytes was investigated on chiral stationary phases based on teicoplanin (Chirobiotic T) and quinidine derivative (Chiralpak QD-AX). Enantioseparation of FMOC-derivatized amino acids was performed in polar- organic separation mode on Chirobiotic T and in reverse-phase and polar-organic separation modes on Chiralpak QD-AX. Both columns were mutually compared. The Chiralpak QD-AX column in combination with polar-organic separation mode (methanol/acetonitrile/acetic acid/ammonium acetate 80/18/2/0.1 (v/v/v/w)) was shown to be the most suitable for separation of mixture of FMOC-amino acids. Under the optimized conditions quantitation of the individual FMOC-D- and FMOC-L-enantiomers, as well as a mixture of FMOC-D- and FMOC-L- enantiomers was performed. Limits of detection ranged from 15.1 to 31.3 ng/ml for FMOC-D-amino acids and from 27.7 to 48.8...
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